Methods and compositions for determining glucose in blood

ABSTRACT

Colorimetric methods and compositions for quantitatively determining the glucose content of blood plasma or serum by heating a deproteinized sample of blood plasma or serum with an alkaline ferricyanide solution, followed by the addition of ferric ions and a 5-(2-pyridyl)-2H-1,4-benzodiazepine or water soluble salts thereof to produce a brilliant purple colored solution which can be quantitated by standard colorimetric means.

United States Patent Klein [451 Apr. 4, 1972 [54] METHODS ANDCOMPOSITIONS FOR DETERMINING GLUCOSEIN BLOOD OTHER PUBLICATIONS Hawk eta1., Practical Physiological Chemistry, 13th ed., Me-

[72] Inventor: Bernard Klein, New Hyde Park, NY. Hill QP514H4, 95 P-567, 575 Assignee Hoffman La Roche I Nu N J Aloe Scientific Co., Catalog103, p. 1011, 1041, 1065, 1073 I nc., ey,

22 Filed; 19, 19 9 Primary Examiner-Morris O. Wolk Assistant Examiner-R.M. Reese [21] APPI'NO: 8 At'torneySamuel L. Welt, Jon S. Saxe, BernardS. Leon,

Gerald S. Rosen and R. Hain Swope [52] US. Cl. ..23/230 R, 23/230 B,252/408,

260/239.3'D ABSTRACT [3;] int. ..C09k 3/2273?! Colorimetric methods andcompositions for quantitatively 1 e o m 2SZ'I4O8 determining the glucosecontent of blood plasma or serum-by heating a deproteinized sample ofblood plasma or serum with [56] R f cted an alkaline ferricyanidesolution, followed by the addition'of e erences l ferric ions and a5(2-pyridyl)-2H-l,4-benzodiazepine or UNITED STATES PATENTS watersoluble salts thereof to produce a brilliant purple colored solutionwhich can be quantitated by standard 3,098,717 7/1963 Ferrari, Jr..23/230 colorimetric means 3,449,081 6/1969 Hughes ..23/253 3,506,4044/1970 Evans et a1. ..23/230 B 11 Claims, 3 Drawing Figures SAMPLE OPLATE O0 O0 SAMPLE ml/min F WM 0,0I5 0.|o

DIALYZER DMC 19.9,;- 0 i N, SALINE 0.100 3140 MIXERS I i -l-@-f- AIR0065 1.60 G; Fe (CN)? 0.!00 3.40

n AIR 0065 L60 A HEATING I U FeCl 0.073 200 BATH H2 I Q REAGENT 0.0m2450 l I 3W6 wAsTE F/C 0.065 1.60 Cg PROPORTIOI'VING I 'Q TUBE SIZEINCHES RECORDER 5B0nm AUTOMATED GLUCOSE ANALYSIS FLOW DIAGRAM PatentedApril 4, 1972 2 Sheets-Sheet 2 SBOnm M FIG. 2

1 I I v 0 50 I00 200 300 40 mg GLUCOSE IOOml E C O (I) \0 1 I l 1 1 l 1l 1' BACKGROUND OF THE INVENTION The need for a quantitatively accuratemethod for the deter- 5 B is selected from mination of glucose in blood,e.g. plasma and serum, using small amounts of specimen, yet which issimple enough to be effectively utilized in the clinical situation andsufficiently economical for mass screening has long been felt. Inaddition, it has been considered most desirable that such a method bereadily adaptable to an automated sequential or continuous flow systemin order that a great many samples may be processed rapidly and with thehighest possible accuracy. There is a need forsuch an automatedsequential or continuous flow of system which is capable of highlyaccurate results before the diagnostic testing of large numbers ofpersons for the incidence of diabetes among them. A simple accuratetest, which is both rapid and reliable, is of great value as an aid inthe detection and treatment of diabetes and as an adjunct to routinescreening operations in clinics and for periodic screening of patientsin hospitals, nursing homes and similar institutrons.

Many techniques have been developed for quantitatively determining theglucose content of blood, plasma or serum. One such technique utilizesthe enzyme glucose oxidase which catalyzes the oxidation of glucose togluconic acid. In the more common test, this enzyme is combined with asubstance having a peroxidative activity which induces the oxidation ofan indicator such as o-toluidine in the presence of hydrogen peroxideformed by the glucose oxidase. This method, though specific, has provedto be too complex, expensive and time consuming for general use.

Other further metric techniques which are adaptable to automatedprocedures have been found to be not sufficiently sensitive for todaysstandards or undesirable in that they require comparatively large volumeof specimen.

The diagnostic compositions and methods of the present invention providea reliable, convenient test for the quantitating of glucose in the bloodas well as affording a method whereby the quantitative determination maybe carried out in a continuous sequential or flow system. Further, thediagnostic compositions and methods of the present invention overcomemany of the disadvantages of the prior art methods of determiningglucose in blood by not requiring a high degree of laboratory skill andtechnology using a small specimen volume, yet being highly accurate inthe clinical situation.

BRIEF SUMMARY OF THE INVENTION In accordance with the invention, a5-(2-pyridyl)-2H-l,4- benzodiazepine or water soluble salt thereofpreferably in combination with a buffer, is added with an aqueoussolution of ferric chloride to deproteinized plasma or serum which hasbeen treated with an aqueous alkaline ferricyanide solution, whereby apurple solution is obtained which can be quantitated as to its glucosecontent by standard colorimetric means.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the invention acompound selected from the group consisting of compounds of the formulawherein A is selected from the group consisting of and and CH,; R isselected from the group consisting of halogen, hydrogen,trifluoromethyl, nitro and amino; R, is selected from the groupconsisting of H R1---Rg 9 hydrogen, lower alkyl and 1 Re C uHzn sistingof hydrogen, hydroxy, lower alkyl, lower alkoxy and lower alkanoyloxy; Ris Z-pyridyl; R is selected from the group consisting of lower alkyl andhydrogen; R is selected and R and R where taken together with theirattached nitrogen atom form a radical selected from the group consistingof piperazinyl, lower alkyl substituted piperazinyl, pyrrolidinyl, loweralkyl substituted pyrrolidinyl, piperidinyl and lower alkyl substitutedpiperidinyl; R is lower alkyl; and R is selected from the groupconsisting of lower alkyl and hydrogen and water soluble salts thereof,preferably in combination with a buffer, is added with an aqueoussolution of ferric chloride to deproteinized blood plasma or serum whichhas been treated with an aqueous alkaline ferricyanide solution, wherebya purple solution is obtained which can be quantitated by standardcolorimetric means.

Examples of benzodiazepine compounds of formula I above which areparticularly suitable as the color-forming reagent in the process ofthis invention include the following:

7-bromo-l ,3-dihydro-l-[4-(4-methyl-l-piperazinyl)butyl]-5-(2-pyridal)-2H-l ,4-benzodiazepin-2-one;

7-aminol ,3-dihydro-5-( 2-pyridyl)-2H-l ,4-benzodiazepin- 2-one;

benzodiazepin-2-one;

2H- 1 ,4-benzodiazepinel -yl)propyl]urea whose preparation is disclosedin US. Pat. No. 3,464,978 issued Sept. 2, 1969;

benzodiazepine;

7-amino-l ,3-dihydrol -methyl-5-( 2-pyridyl)- l H- l ,4-

benzodiazepine;

7-bromol ,3-dihydro-( 3-dimethylaminopropyl)-5-( 2- pyridyl)-2H- l,4-benzodiazepin-2-one;

7-bromol ,3-dihydro-5-(2-pyridyl)-2I-I-l ,4-benzodiazepin-2-one-4-oxide;

7-bromol ,3-dihydro-5-( 2-pyridyl)-2H- l ,4-benzodiazepin- 2-one;

7-bromol ,3-dihydrol B-hydroxypropyl)-2-( 2-pyridyl 2H- 1,4-benzodiazepin-2-one; and

7-bromo-5-( 2-pyridyl)-l ,3.-dihydrol 3-( N- cyanomethylamino)propyl]2I-I- 1 ,4-benzodiazepin-2-one whose preparation is disclosed inUS. Pat. No. 3,464,978.

The term lower alkyl as used throughout this specification includes bothstraight and branched chain alkyl groups having from one to seven carbonatoms such as methyl, ethyl, propyl, isopropyl and the like. The termlower alkanoyloxy" refers to both straight chain and branched chainaliphatic carboxylic acid moieties such as acetoxy, propionyloxy,butyryloxy and the like. The term halogen includes bromine, chlorine,fluorine and iodine. Also included within-the purview of the presentinvention are the water soluble acid addition salts of the compounds offormula I above. Any conventional water soluble acid addition salts ofthe compounds of formula I above may be utilized in the process of thisinvention to quantitatively determine the iron content of aqueoussolutions. Among the acid addition salts which can be utilized inaccordance with this invention, includes salts of compounds of theformula I with organic or inorganic acids such as hydrochloric acid,hydrobromic acid, nitric acid, sulfuric acid, acetic acid, formic acid,succinic acid, maleic acid, ptoluenesulfonic acid and the like.

The color differentiation with varying concentrations of ferrous ionsproduced by the compound of formula I above is such that theconcentration of ferrous ions produced by the instant diagnostic reagentcomposition in situ can easily be determined by standard colorimetricinstruments. Furthermore, the compounds of formula I are not sensitiveto extraneous sources and therefore are not affected by tracecontaminants. The method of this invention provides a simplecolorimetric means for quantitatively determining the glucose content ofblood plasma and serum.

In accordance with the present invention the glucose content of bloodplasma or serum can be determined by first heating a deproteinizedsample with an aqueous solution containing ferricyanide ions to form anaqueous solution containing gluconic acid and ferrocyanide ions, coolingthe solution and adding an aqueous solution containing ferric ions and acompound of formula I above wherein ferricyanide and ferrous ions areformed and the ferrous ions thus produced react with the compound offormula I, preferably in the presence of a buffer, to produce abrilliant deep purple color and colorimetrically quantitating the amountof glucose present in the sample. This procedure provides a simple andquick method for quantitatively determining the glucose content of ablood sample which is ideally suited for routine diagnostic use.

In accordance with the present invention, the specimen to be tested isinitially treated with a conventional neutral deproteinizing agent suchas, for example, an aqueous solution of either sodium or bariumhydroxide and zinc sulfate, or an acidic deproteinizing agent such as,for example, tungstic acid or trichloroacetic' acid. Of these, tungsticacid or an aqueous solution of barium hydroxide and zinc sulfate arepreferred. The specimen is well mixed with the deproteinizing agent andcentrifuged at high speed to obtain a clear supemate. A 0.1 ml. aliquotof the superna'te is then heated to from 90 C. to about 100 C.preferably about 95 C. with 2.0 ml. of an aqueous alkaline solutioncontaining ferricyanide ions. The mixture is rapidly cooled after about5 minutes heating and treated with 2.0 ml. of an aqueous solutioncontaining ferric ions such as, for example, ferric chloride, and 2.0ml. of an aqueous solution of a compound of the formula I. The solutionsare mixed and the absorbance of the violet blue color which developsover about 10 minutes is measured at 580 nm against both a standardglucose solution similarly treated and a reagent blank.

The solution containing ferricyanide ions can be made from any watersoluble ferricyanide salt which does not otherwise interfere with thereaction such as, for example, potassium ferricyanide and sodiumferricyanide. Potassium ferricyanide is preferred in the practice of thepresent invention. This reagent may be made in quantity if so desiredand used as needed. The

appropriate amount of potassium ferricyanide is dissolved in an aqueousalkaline medium'such as, for example, a 2 percent sodium carbonatesolution. The quantity of ferricyanide salt utilized in preparing thereagent is variable. However, a sufficient quantity must be utilized toreact with all the glucose in the specimen to furnish a positiveindication of elevated glucose blood levels when the diagnostic methodof the present invention is being utilized as a diagnostic or a massscreening tool.

Generally, it is preferred that for each ml. of blood plasma or serumtested, the reagent solution contain from about l.8 x 10" moles to about7.0 X 10 moles of ferricyanide salt, preferably from about 3.5 X 10"moles to about 5.0 X 10" moles per ml. of plasma or serum utilized.

The quantity of ferric ions added to the sample ferricyanide ion mixtureis again variable. However, it is preferred to utilize a quantity offerric ion slightly in excess of the molar quantity of ferricyanide ionsadded to the sample. The utilization of such an excess insures thatthere will be sufficient ferric ions present to react with theferrocyanide ions generated by the initial reaction between theferricyanideions and the glucose in the sample. The ferric ions may besupplied as any water soluble ferric salt which does not interfere withthe diagnostic determination such as, for example, ferric chloride,ferric nitrate, ferric sulfate and the like. Of these, ferric chlorideis preferred.

The quantity of the compound of formula I which is added to the aqueousmixture is variable. In all instances, however, there must be asufficient quantity of the compound of formula I present to react withall of the ferrous ions generated bythe reaction between the ferric ionsand the ferrocyanide ions. This quantity is most conveniently determinedby equating the quantity of the compound of formula I with that of theferric ions to insure the stoichiometry of the chelation reaction.

It is preferred to maintain the test medium at a pH of about 4.0 toabout 5 .0, preferably about 4.5. This can most easily be accomplishedby adding suitable buffers to the ferric ion reagent and the reagentcontaining the compound of formula I. Buffering these reagents alsomakes them stable in aqueous solution when they are made up in quantityfor large scale laboratory testing.

In general, any recognized buffer pair suitable for the maintenance ofsuch a pH range as described above can be utilized.

Preferably, there can be utilized as a buffer pair a water soluble saltof acetic acid and acetic acid. Of the water soluble salts of aceticacid sodium acetate is preferred. However, ammonium acetate, potassiumacetate or other water soluble salt of acetic acid can be used, ifdesired. Although the quantities of the buffer pair comprising a watersoluble acetic acid salt and acetic acid are variable, the presentinvention contemplates the use of a sufficient quantity of theacidcomponent, e.g. acetic acid, to provide a final test sample having apH in the range of from about 4.5 to about 5.5. By final test sample ismeant a solution containing the ferricyanide ions, the ferric ions andthe benzodiazepine color reagent. In general, there is contemplated thepreparation of a solution of both the ferric ions and the benzodiazepinecolor former which contains per liter about 1.0 mole of a water solublesalt of acetic acid to about 1.0 to about 2.0 moles of acetic acid.

From the foregoing description it is evident that thecompositions of thepresent invention may be utilized or handled as prepared aqueous stocksolutions, aqueous concentrates or in a dry powder form. In either theconcentrate or the powder form, sufficient buffering agents are added tostabilize the compositions when the working dilutions are made andmaintain the pH of the reaction mixture at between 4.5 and 5.5preferably about 4.8.

In utilizing the compositions of the present invention, the addition ofthe compound of formula I to the test system immediately produces thedesired purple coloration. The color deepens as the reaction proceeds tocompletion. The reaction mixture ceases to undergo anycolor changesdiscernible to the naked eye after it has been allowed to stand for ashort time at room temperature. Accordingly, in order to insure uniformcoloring, the aqueous solution should be aliowed to stand until itscolor appears to have become constant. In general, it has been foundthat the full development of the purple color will occur over a periodof from about 5 to minutes after the addition of the compound of formulaI. In most cases 10 minutes is a sufficient period of time to allow forfull color development.

The quantitation of the glucose in the colored sample can be carried outby any conventional colorimetric method utilizing standardspectrophotometers such as a Beckman Spectrophotometer, ColemanSpectrophotometer and the like.

The principle of the diagnostic method according to the presentinvention is based on a series of coupled reactions. Initially, glucosepresent in the sample undergoing analysis reduces the ferricyanide ionin the added first reagent to ferrocyanide ions, in turn formferricyanide ions and ferrous ions with the addition of the secondreagent which comprises a source of ferric ions such as, for example,ferric chloride, a buffer and a compound of the formula I. The ferrousions thus generated react with the compound of the formula I to producea brilliant deep purple color. The purple color is thereaftercolorimetrically measured and the glucose content of the samplequantitatively determined.

The quantitative determination of the glucose content in a specimen iscarried out as follows: the optical density of the purple colordeveloped in the sample by the method of the present invention ismeasured against a reagent blank at 580 nm utilizing a standardspectrophotometer such as, for example, a Coleman Spectrophotometer,employing a cuvette with a 19 mm. light path. The quantity of glucose inthe specimen is determined in the conventional manner from theabsorbance of the specimen with reference to the absorbance of the colorproduced by a glucose standard similarly treated. The glucose content ofthe specimen is calculated in accordance with the following formula:

Glucose content of specimen (mg./ 100 ml.)

Absorbance of specimen Absorbance of standard As indicated heretofore,the present invention provides an extremely important diagnostic tool.In addition, the method of the present invention affords a rapid andaccurate determination of the glucose content of body fluids such asplasma or serum with results that are characterized by a high degree ofreproducibility.

In another aspect of the present invention, the analytical compositionsas described are utilized in a method of analyzing the glucose contentof body fluids automatically by discrete sequential sampling or bycontinuous flow apparatus. The latter method consists essentially ofmixing specimens in continuous flow with normal saline, dialyzing themixture to produce an aqueous protein-free solution containing theglucose, mixing the aqueous solution with an aqueous alkaline solutioncontaining ferricyanide ions, passing the resulting mixture through aheating bath to raise the temperature thereof to about95 C., mixing theheated aqueous solution with an aqueous solution of a ferric salt and acompound of the formula I at a constant pH of from about 4.5 to about5.5 and passing the resulting solution through an apparatus whichquantitatively determines the glucose content thereof photometrically.

FIG. 1 is a schematic flow diagram illustrating a continuous flowautomated system for analyzing glucose in biological fluids utilizingthe diagnostic composition of the present invention.

FIG. 2 is a recording of the photometric response obtained whenutilizing the automated system of FIG. 1.

FIG. 3 is a plot in terms of absorbance of the photometric responseillustrated in FIG. 2.

In FIG. 1, a continuous flow automated testing system is,

showriglgrnati cally wherein a specimen sample to be tested, i.e. serumor plasma, is drawn up in sequence from separate sample cups inthesample plate which rotates at a constant speed to provide the systemwith 20-60 specimen samples with a 2:1 wash ratio per hour. A sample, sodrawn, is mixed in flow with normal saline and passed through a glassmixing coil of conventional design. After the mixture has passed throughthe mixing coil, it is next pumped through a dialyzer module that isprovided with a cellophane membrane or the like through which theglucose passes in aqueous solution by dialysis. The dialyzer module ismaintained at a constant temperature of 37 C. The residual,non-diffusable portion of the sample is discarded. As the aqueousglucose solution passes through the dialyzer module membrane it isadmixed with an aqueous alkaline solution containing ferricyanide ions,preferably in the form of potassium ferricyanide, the glucose and theferricyanide ions are passed in solution through a heating bath whichraises the temperature of the mixture to C. As this passage takes placethe glucose and ferricyanide ions are reacting to form gluconic acid andferrocyanide ions. The heated aqueous stream is then mixed in continuousflow with an aqueous solution containing ferric ions, preferably in theform of ferric chloride, and a reagent stream comprising the5-(2-pyridyl)-2H-l ,4-benzodiazepine color reagent of formula I. Thecolor reagent, preferably 7-bromo-l,3-dihydro-l-3-dimethylaminopropyl)-5-( 2-pyridyl)-2I-I- l ,4- benzodiazepine-2-oneis maintained at a pH of about 4.5 to 5.5, preferably at about 5.0. Themixture is then passed through a second mixing coil. As the mixture isin transit through this coil, the ferric ions and ferrocyanide ionsreact to form ferricyanide ions and ferrous ions which in turn reactwith the benzodiazepine color reagent to form a brilliant purplecoloration. Photometric measurements are then performed at 580 nm in a15 mm. flow-cell colorimeter, i.e., the absorbance of the solution to betested is measured at 580 nm in a flow-cell colorimeter using a 580 nmfilter. The results of the colorimetric readings are recorded on aconventionalrecording mechanism.

The continuous flow system illustrated in FIG. 1 aspirates at a rate of20 to 60 specimens/hour. The rate of flow in ml./min. of the materialsentering the system according to a preferred technique is illustrated inFIG. 1. The materials entering the system are pumped into it by anysuitable pumping means adjusted to maintain the rate of flow illustratedin FIG. 1. The mechanism for the system of the present invention can beconveniently provided by a manifold assembly prepared in accordance withthe system illustrated in FIG. 1 adaptable to the TechniconAutoanalyzer.

In FIG. 2 the absorbance of solutions containing graduated amounts ofglucose, e.g. 50 mg./ ml., 100 mg./ 100 ml. etc. are plotted as a graphagainst concentration.

In FIG. 3 the photometric response of solutions containing differentconcentrations of glucose is demonstrated. The drawing illustrates fourseparate experiments, each of which represents passage through theautomated system of FIG. 1 of a sequence of at least three solutionshaving glucose concentrations in the order of low to high to low, suchas, for example, 50 mg. per 100 ml. to 400 mg. per 100 ml. to 50 mg. per100 ml. These experiments were conducted to illustrate the sensitivityof the automated system. The difference in the response curve forsimilar concentration sequences represents a variance in the speed withwhich they were passed through the system.

The reagents utilized in connection with the automated procedure ofglucose determination comprise aqueous solutions of a ferricyanidereagent, a ferric ion containing reagent and and the buffered colorforming reagent. The ferricyanide reagent comprises sufficientferricyanide to react with all the glucose in the sample, for example,0.1 I5 g. potassium ferricyanide dissolved in 1 liter of 0.05 percentsodium hydroxide and 0.9 percent sodium chloride. The ferric ioncontaining solutions comprises suflicient ferric ions to react with allthe ferrocyanide ions formed in the initial reaction, for example, 0. 27 g. ferric chloride dissolved in 1 liter of distilled water andbuffered to a pH of about 4.5 with a sodium acetate/acetic acid buffercouple. The color-forming reagent comprises sufficient color-formingcompound to react with the ferrous in the ions formed by the reaction ofthe ferric ions and the ferrocya nide ions, for'example, 2.0 g. of acompound of formula I, 82.0 g. of ammonium acetate and approximately60.0 ml. of glacial acetic acid in a liter of distilled water. The pH ofthe solution is maintained between about 4.4 and 4.6.

In the practice of the invention according to the automated procedure,iron-free distilled water is pumped through the system for 10 minutes.The system is then switched to reagent and the pumping is continueduntil a steady base line is obtained on the recorder chart. The baseline is set to 0.01A (95 percent transmission).

The standards in the sample tray are aspirated at a rate of 20 to 60'(2:1 wash ratio) samples per hour. The specimens to be analyzed are thensampled, with a standard glucose specimen which is aspiratedintermittently to insure qualitative control. p The glucose content ofeach specimen is determined by reference to a calibration curve preparedby plotting the corrected absorbances of the glucose standards againstconcentrations in mg./ 100 ml. Table l sets forth a comparison ofresults obtained when 10 randomly selected plasma specimens wereanalyzed utilizing the automated and manual glucose procedures of thepresent invention.

TABLE I Comparison of Manual and Automated Glucose Analysis (mg/100 ml.)

Specimen No. Manual Automated Difference In Table II, the recovery ofglucose added to pooled serum aliquots is given. An average recovery of99.3 percent (range 94.2-l03.5 percent was realized.)

TABLE II.--RECOVERY OF GLUCOSE ADDED TO SERUM For a fuller understandingof the nature and objects of the present invention, reference may be hadto the following examples which are given merely as furtherillustrations of the invention and are not to be construed in a limitingsense.

EXAMPLE 1 To a stirred solution off 22.0 g. of 7-bromo-l,3-dihydro-5-(2-pyridyl)-2H l,4-benzodiazepin 2-one in 55.0 ml. of dryN,N-dimethylformamide was treated with 11.0 ml. of a methanolic solutionof sodium methoxide (0.0835 mole of 75 4 EXAMPLE 2 The 7-bromol,3-dihydrol-( 3-dimethylaminopropyl )-5 2 was dissolved in sufficientmethanol to provide a 10 percent solution. This solution was thensaturated with hydrogen chloride. A sufficient amount of ether was addedto cause turbidity. The resultant mixture was allowed to cool forseveral hours. 7-Bromol ,3-dihydrol 3-dimethylaminopropyl)-5-( 2 9y y 2H1L .-.l2=nm iiaz9p n: 1e d ydr9.h. 2n'ds precipitated out on standingand was separated by filtration. The salt was recrystallized from amethanol-ether mixture as pale yellow prisms, MP. l8l-l 83 dec.

EXAMPLE 3 This example demonstrates the applicability of the test methodto either blood serum or plasma.

In the method, aliquots of plasma or serum were added in 0.1 ml.quantities to 2.4 ml. of a tungstic acid solution prepared by mixing onevolume 10 percent sodium tungstate and 8 volumes N/ l2 sulfuric acid.Each mixture was well mixed and then centrifuged at 2,500 rpm. for ISminutes. A 0.1 ml. aliquot of the clear supernatant liquid was treatedwith 2.0 ml. of a ferricyanide solution which had been prepared bydissolving 0.1 15 g. of potassium ferricyanide in one liter of a 2percent aqueous solution of sodium carbonate. ,The deproteinized fluidferricyanide mixture was heated for 5 minutes in a boiling water bath,rapidly cooled to about 25 C. and treated with 2.0 ml. of a ferric ionreagent prepared by dissolving 0.27 g. ferric chloride hexahydrate inone liter of an acetate buffer comprising 272.0 g. sodium acetate and294.0

- ml. glacial acetic acid, and 2.0 ml. of a solution prepared bydissolving 2.0 g. of the compound produced in Example 2 in one liter of1M acetate bufl'er. The solutions were thoroughly mixed and theabsorbance of the violet blue color that develops was measured afterabout 10 minutes against a reagent blank at 580 nm in a ColemanSpectrophotometer using a cuvette with a 19 mm. light path.

The glucose content of the specimens was obtained by reference to acalibration curve prepared by plotting the absorbances (A) given bystandard glucose solutions treated in the same manner againstconcentration or by the Beer-Lambert formula. Utilizing 4.0 mcg.glucose/0.1 ml. as a standard, the concentration of the specimen wascalculated according to the formula:

(Absorbance of Specimen/Absorbance of Standard) x l00 mg. glucose/ ml.

For comparative purposes, glucose analyses were also conducted on a likenumber of samples utilizing a modified Folin- Wu procedure as describedby B D. Tonks in American Journal of Clinical Pathology, 22:1009,(l952).

The results obtained utilizing the aforesaid two techniques 2? 595.1%?!thef ll w s il Benzediazeplne test Folin-Wu Difference Specimen (mg.glucose/ (mg. glucose] (mg. glucose] Difference,- number 100 ml.) 100ml.) 100 ml.) percent The comparative data obtained utilizing plasmaspecimens was completely analogous to that with serum.

Utilizing similar reagents and quantities as were employed in Example 3,tests were conducted utilizing the compound prepared in Example 2 andcomparing the results with the glucose content deproteinized plasmaprepared as described in B. Klein in Clinical Chemistry, 5; 62, (1959)and designated Somogyi filtrates.

The results obtained are set forth in the following table:

In an analogous manner to that employed in Examples 3 and 4, serumspecimens from 20 randomly selected blood samples obtained from healthyindividuals and hospitalized patients were tested for glucose content.For comparative purposes, the glucose content of the same blood samplewas also determined utilizing a standard glucose oxidase procedure. Theglucose oxidase procedure employed herein is described in detail by R.Richterich and J. P. Colombo in Klin. Woch., 40, 1208, (1962) and A.Saifer and S. Gerstenfeld in J. Lab. Clin. Med., 51, 448, (1958).

The results obtained utilizing the two techniques are set forth in thefollowing table:

Comparison of Glucose Analyses (milligrams glucose/100 ml.)

1. A process for quantitating the glucose content of blood serum orplasma comprising:

a. treating a deproteinized sample of blood serum or plasma with anaqueous alkaline solution containing a source of ferricyanide ions;

b. heating the mixture from step (a) to a temperature of from about 90C.to about l00C.;

c. adding to the mixture from step (b) a source of ferric iron ions;

d. reacting the mixture of step (c) with a benzodiazepine compoundselected from the group of the compounds of the formula wherein A isselected from the group consisting of and B is selected from the groupof hydrogen, lower alkyl and C uHzn rt is an integer from 2 t o 7 R3 isselected from the group consisting of hydrogen, hydroxy, loweralkylIlower allioxy a nd lower alkanoyloxy; R is Z-pyridyl; R isselected from the group consisting of lower alkyl, hydrogen,

and

GEN

gnd R and "R6, where taken together with their attached nitrogenatom,from a radical selected from the group consisting of piperazinyl, loweralkyl substituted piperazinyl, pyrrolidinyl, lower alkyl substitutedpyrrolidinyl, piperidinyl, and lower alkyl substituted piperidinyl; R islower alkyl; and R is selected from the group consisting of lower alkyland hydrogen, and water soluble acid addition salts thereof; and

e. colorimetrically quantitating the glucose present by means of saidcolor.

2. The process in accordance with claim 1 wherein said source of ferricions consists essentially of an aqueous solution of ferric chloridebuffered to a pH of between from about 4.0 to about 5.0.

3. The process in accordance with claim 1 wherein said benzodiazepinecompound is added as an aqueous solution buffered to a pH of from about4.0 toabout 5.0.

4. The process in accordance with claim 1 wherein said benzodiazepinecompound is selected from the group consisting of 7-bromo-l,3-dihydro-l-( 3-dimethylaminopropyl)-5-(2-pyridyl)-2H-l,4-benzodiazepin-2-one and water soluble acid additionsalts thereof.

1 1 l2 5. A process for quantitating the glucose content of blood 7. Theprocess in accordance with claim 5 wherein said j serum or plasmacomprising: buffer present in the aqueous solution containing ferric a.treating a deproteinized sample of blood serum or plasma chloride andthe aqueous solution containing the with an aqueous alkaline solutioncontaining a source of benzodiazepine compound is a buffer paircomprising a water ferricyanide ions; 5 soluble salt of acetic acid andacetic acid.

b. heating the mixture from step (a) to a temperature of 8 A method forthe quantitative analysis of the glucose confrom about 90C. to about100C; tent of blood plasma or serum consisting essentially of providc.adding tov the mixture of step (b) an aqueous solution ing in continuousflow, the sequential steps comprising:

containing ferric chloride and abuffer; a. combining in continuous flowa measured specimen of d. adding to the mixture of step (c) an aqueoussolution 10 plasma or serum with an isotonic solution of sodiumcontaining a buffer and a benzodiazepine compound chloride; selectedfrom the group consisting of compounds of the b. passing said mixturethrough a separating zone, thereby formula separating by dialysis insaid zone from said mixture a clear aqueous solution; c. mixing saidclear aqueous solution with a reagent com- N-B H prising an alkalineaqueous solution of a water soluble ferricyanide salt; R, d. passingsaid aqueous mixture through heating means thereby raising thetemperature thereof to from about A I 95C. to about iooc.;'

e. mixing said heated aqueous mixture by concurrent flow h i A iselected from the group consisting of with a first reagent comprising abuffered aqueous solution of a ferric iron salt and a second reagentcomprising a buffered, aqueous solution of a color-forming compound \mselected from the group consisting of compounds of the i l formula andR2 N-B H R; 0 B is selected from the group consisting of A/ 3 o wt, Iwherein A is selected from the group consisting of and Cl-l,; R isselected from the group consisting of 1 halogen, hydrogen,trifluoromethyl, nitro, and amino; R, is selected from the groupconsisting of 40 and H C='N r; R1 Rs 4 \O r H B is selected from thegroup consisting of hydrogen, lower alkyl and 0 Rs -H o HhN and --CH,--;R, is selected from the group consisting of RB halogen, hydrogen,trifluoromethyl, nitro, and amino; R, is

selected from'the group consisting of n is an integer from 2 to 7; R; isselected from t h e group consisting of hydrogen, hydroxy, lower alkyl,lower allioxy and H lower alkanoyloxy; R, is 2-pyridyl; R is selectedfrom the group consisting of lower alkyl; hydrogen,

fiNH2 hydrogen, lower alkyl and and s GEN CnH2nN jl ld R and R wheretaken together with their attached nitrogen atom, fonn a radicalselected from the group consist- M ing of piperazinyl, lower alkylsubstituted piperazinyl, pyrn is an integer from 2 to 7; R is selectedfrom the gr o gn rolidinyl, lower alkyl substituted pyrrolidinyl,piperidinyl, and sisting of hydrogen, hydroxy, lower alkyl, lower alkoxyand lower alkyl substituted piperidinyl; R is lower alkyl; and R, islower alkanoyloxy; R is 2-pyridyl; R is selected from the selected fromthe group consisting of lower alkyl and group consisting of lower alkyl;hydrogen, hydrogen and water soluble acid addition salts thereof; and

e. colorimetrically quantitating the glucose present by means of saidcolor.

6. The process in accordance with claim 5 wherein said source offerricyanide ions is potassium ferricyanide. and

GEN

; and R and R;, where taken together with their attached nitrogen atom,form a radical selected from the group consisting of piperazinyl, loweralkyl substituted piperazinyl, pyrrolidinyl, lower alkyl substitutedpyrrolidinyl, piperidinyl, and

lower alkyl substituted piperidinyl; R is lower alkyl; and R is selectedfrom the group consisting of lower alkyl and hydrogen thereof and watersoluble acid addition salts thereof thereby forming a colored solution;and

f. flowing said colored solution to an analyzing zone andphotometrically determining quantitatively the amount of glucose presentduring the flow of said colored solution through said analyzing zone.

9. The method in accordance with claim 8 wherein said first reagent andsaid second reagent are buffered to a pH of from about 4.0 to about 5.0with a buffer pair comprising a water soluble salt of acetic acid andacetic acid.

10. The method in accordance with claim 8 wherein said colorformingcompound is selected from the group consisting of 7-bromo-l ,3-dihydrol3-dimethylaminopropyl)-5-( 2- pyridyl)-2H-1,4-benzodiazepin-2-one andwater soluble acid addition salts thereof.

11. The method in accordance with claim 8 wherein said water solubleferricyanide salt is potassium ferricyanide and said ferric iron salt isferric chloride.

2. The process in accordance with claim 1 wherein said source of ferricions consists essentially of an aqueous solution of ferric chloridebuffered to a pH of between from about 4.0 to about 5.0.
 3. The processin accordance with claim 1 wherein said benzodiazepine compound is addedas an aqueous solution buffered to a pH of from about 4.0 to about 5.0.4. The process in accordance with claim 1 wherein said benzodiazepinecompound is selected from the group consisting of7-bromo-1,3-dihydro-1-(3-dimethylaminopropyl)-5-(2-pyridyl)-2H-1,4-benzodiazepin-2-one and water soluble acid addition salts thereof. 5.A process for quantitating the glucose content of blood serum or plasmacomprising: a. treating a deproteinized sample of blood serum or plasmawith an aqueous alkaline solution containing a source of ferricyanideions; b. heating the mixture from step (a) to a temperature of fromabout 90* C. to about 100*C.; c. adding to the mixture of step (b) anaqueous solution containing ferric chloride and a buffer; d. adding tothe mixture of step (c) an aqueous solution containing a buffer and abenzodiazepine compound selected from the group consisting of compoundsof the formula wherein A is selected from the group consisting of and Bis selected from the group consisting of and -CH2-; R1 is selected fromthe group consisting of halogen, hydrogen, trifluoromethyl, nitro, andamino; R2 is selected from the group consisting of , hydrogen, loweralkyl and ; n is an integer from 2 to 7; R3 is selected from the groupconsisting of hydrogen, hydroxy, lower alkyl, lower alkoxy and loweralkanoyloxy; R4 is 2-pyridyl; R5 is selected from the group consistingof lower alkyl; hydrogen, and ; and R5 and R6, where taken together withtheir attached nitrogen atom, form a radical selected from the groupconsisting of piperazinyl, lower alkyl substituted piperazinyl,pyrrolidinyl, lower alkyl substituted pyrrolidinyl, piperidinyl, andlower alkyl substituted piperidinyl; R7 is lower alkyl; and R8 isselected from the group consisting of lower alkyl and hydrogen and watersoluble acid addition salts thereof; and e. colorimetricallyquantitating the glucose present by means of said color.
 6. The processin accordance with claim 5 wherein said source of ferricyanide ions ispotassium ferricyanide.
 7. The process in accordance with claim 5wherein said buffer present in the aqueous solution containing ferricchloride and the aqueous solution containing the benzodiazepine compoundis a buffer pair comprising a water soluble salt of acetic acid andacetic acid.
 8. A method for the quantitative analysis of the glucosecontent of blood plasma or serum consisting essentially of providing incontinuous flow, the sequential steps comprising: a. combining incontinuous flow a measured specimen of plasma or serum with an isotonicsolution of sodium chloride; b. passing said mixture through aseparating zone, thereby separating by dialysis in said zone from saidmixture a clear aqueous solution; c. mixing sAid clear aqueous solutionwith a reagent comprising an alkaline aqueous solution of a watersoluble ferricyanide salt; d. passing said aqueous mixture throughheating means thereby raising the temperature thereof to from about 95*C. to about 100* C.; e. mixing said heated aqueous mixture by concurrentflow with a first reagent comprising a buffered aqueous solution of aferric iron salt and a second reagent comprising a buffered, aqueoussolution of a color-forming compound selected from the group consistingof compounds of the formula wherein A is selected from the groupconsisting of and ; B is selected from the group consisting of and-CH2-; R1 is selected from the group consisting of halogen, hydrogen,trifluoromethyl, nitro, and amino; R2 is selected from the groupconsisting of , hydrogen, lower alkyl and ; n is an integer from 2 to 7;R3 is selected from the group consisting of hydrogen, hydroxy, loweralkyl, lower alkoxy and lower alkanoyloxy; R4 is 2-pyridyl; R5 isselected from the group consisting of lower alkyl; hydrogen, and ; andR5 and R6, where taken together with their attached nitrogen atom, forma radical selected from the group consisting of piperazinyl, lower alkylsubstituted piperazinyl, pyrrolidinyl, lower alkyl substitutedpyrrolidinyl, piperidinyl, and lower alkyl substituted piperidinyl; R7is lower alkyl; and R6 is selected from the group consisting of loweralkyl and hydrogen thereof and water soluble acid addition salts thereofthereby forming a colored solution; and f. flowing said colored solutionto an analyzing zone and photometrically determining quantitatively theamount of glucose present during the flow of said colored solutionthrough said analyzing zone.
 9. The method in accordance with claim 8wherein said first reagent and said second reagent are buffered to a pHof from about 4.0 to about 5.0 with a buffer pair comprising a watersoluble salt of acetic acid and acetic acid.
 10. The method inaccordance with claim 8 wherein said colorforming compound is selectedfrom the group consisting of7-bromo-1,3-dihydro-1-(3-dimethylaminopropyl)-5-(2-pyridyl)-2H-1,4-benzodiazepin-2-one and water soluble acid addition salts thereof. 11.The method in accordance with claim 8 wherein said water solubleferricyanide salt is potassium ferricyanide and said ferric iron salt isferric chloride.